Utilization of microRNAs and their regulatory functions for improving biotic stress tolerance in tea plant [Camellia sinensis (L.) O. Kuntze]

Figures & data

Figure 1. The model of miRNA biogenesis and functions in plants. RNA polymerase II (RNA Pol II), primary transcripts (pri-miRNAs), stem-loop precursor miRNAs (Pre-miRNAs), DICER-LIKE-1 RNase III endonucleases (DCL1), double-stranded RNA (dsRNA) binding protein, HYPONASTIC LEAVES1 (HYL1), C2H2-zinc finger protein SERRATE (SE), s-adenosyl methionine-dependent methyltransferase HUA ENHANCER 1 (HEN1), exportin-5 homology protein (HASTY), small RNA degrading nuclease (SDN) class of exonucleases, RNA induced silencing complex (RISC), argonaute family protein (AGO1).

Table 1. Potential target transcripts of E. oblique-induced stress-responsive miRNAs in C. sinensis reported by Jeyaraj et al. (2017b).

miRNAs	Targets	Transcript ID	Score	Range	Biological process
csn-miRn19	Leucine-rich repeat receptor-like serine/threonine-protein kinase BAM3	76159_gi319848380	4	166–189	Cell differentiation
	Transcription factor bHLH63	comp96827_c0_seq8	3.5	805–828	Regulation of flower development
	Probable serine/threonine-protein kinase WNK11	comp100561_c1_seq3	4	39–62	Serine/threonine kinase activity
csn-miRn36	Transcription factor VIP1	comp104613_c3_seq1	4	1524–1547	Defence response
csn-miRn57	Ethylene-responsive transcription factor ERF114	comp104355_c1_seq1	4	43–66	Defence response
	NAC domain-containing protein 72	comp107062_c2_seq1	3	14–37	Regulation of transcription
	NAC domain-containing protein 19	comp107062_c2_seq4	3	14–37	Response to water deprivation
	Probable receptor-like protein kinase	comp107842_c0_seq15	4	5–28	Protein kinase activity
	Uncharacterized calcium-binding protein	comp97730_c1_seq34	2	137–159	Response to cold
	Probable LRR receptor-like serine/threonine-protein kinase	comp99023_c0_seq2	4	54–77	Serine/threonine kinase activity
	Probable serine/threonine-protein kinase WNK11	comp100561_c1_seq3	4	21–45	Serine/threonine kinase activity
csn-miR169b	Nuclear transcription factor Y subunit A-6	24872_gi400010701	3.5	491–514	Regulation of transcription
	Nuclear transcription factor Y subunit A-1	76159_gi319883397	4	1171–1194	Regulation of transcription
	Nuclear transcription factor Y subunit A-9	comp97629_c0_seq16	4	1093–1116	Regulation of transcription
	Nuclear transcription factor Y subunit A-3	comp99688_c0_seq2	3.5	785–807	Regulation of transcription
csn-miRn190	Mitogen-activated protein kinase kinase kinase 13-A	comp107832_c2_seq2	4	4798–4821	Protein autophosphorylation
csn-miRn301	Transcription factor bHLH112	comp99860_c1_seq8	4	650–673	Regulation of transcription
csn-miRn335	Probable glutathione S-transferase	24872_gi400016573	4	410–433	Auxin mediated signalling
	Ethylene-responsive transcription factor ERF114	comp104355_c1_seq1	4	35–57	Ethylene mediated signalling
	Serine/threonine-protein kinase PEPKR2	comp97495_c0_seq1	4	1676–1700	Serine/threonine kinase activity
csn-miRn387	Cysteine-rich receptor-like protein kinase 8	76159_gi319852408	3	139–161	Protein kinase activity
	Cysteine-rich receptor-like protein kinase 7	76159_gi319873174	3	488–510	Protein kinase activity
csn-miRn392	Transcription factor bHLH63	comp96827_c0_seq8	4	783–806	Regulation of flower development
	Transcription factor bHLH112	comp99860_c1_seq8	2.5	650–673	Regulation of transcription
csn-miRn421	Transcription factor bHLH79	comp106187_c7_seq22	4	1256–1279	Regulation of transcription
	Transcription factor bHLH78	comp106187_c7_seq18	4	2332–2355	Regulation of transcription
	LRR receptor-like serine/threonine-protein kinase ERECTA	comp106849_c1_seq5	4	1148–1171	Plant-type cell wall organization
	Putative cysteine-rich receptor-like protein kinase 31	comp107702_c0_seq1	3	132–155	Protein kinase activity
	Transcription factor bHLH123	comp99860_c1_seq9	3	846–868	Regulation of transcription
	Transcription factor bHLH112	comp99860_c1_seq2	3	846–868	Regulation of transcription

Figure 2. The hypothetical model of regulatory networks of Ectropis oblique stress-responsive miRNA in tea plant. Leucine-rich repeat receptor-like protein kinase (LRR-RLKs), Cysteine-rich receptor-like protein kinase (CRR-RLKs), serine/threonine protein kinase (Ser/Thr kinase), glutathione S-transferase (GST), calcium-binding protein (CBP), mitogen-activated protein kinase (MAPKs), basic helix-loop-helix (bHLHs) transcription factor, ethylene-responsive transcription factor (ERFs), basic leucine zipper (bZIP) transcription factor (VIP1), NAC domain transcription factor (NACs) and Nuclear transcription factor Y subunit A (NF-YAs).

Table 2. Potential target transcripts of C. gloeosporioides-induced stress-responsive miRNAs in C. sinensis reported by Jeyaraj et al. (2019).

miRNAs	Targets	Transcript ID	Score	Range	Biological process
csn-miR156a-p3_1ss10TA	LRR receptor-like serine/threonine-protein kinase FLS2	Unigene31494_All	4	621–640	Protein phosphorylation
csn-miR160a, csn-miR160a_L + 1, csn-miR160b, csn-miR160 c	Auxin response factor 15-like isoform X1	CL3788.Contig1_All	1	1224–1244	Regulation of transcription
	Auxin response factor 5-like	Unigene27869_All	0.5	455–475	Regulation of transcription
	Auxin response factor 7-like	CL3788.Contig2_All	1	1554–1574	Regulation of transcription
	Auxin response factor 3	CL7735.Contig3_All	1	1332–1352	Regulation of transcription
	Auxin response factor 1	CL7735.Contig4_All	1	1051–1071	Regulation of transcription
csn-miR164a, csn-miR164a_L + 1_1/2	NAC domain-containing protein 89-like	Unigene18223_All	1.5	849–869	Regulation of transcription
csn-miR166e-p5_2ss10GT17CT	Peroxidase 72-like	CL2569.Contig1_All	4	106–123	Response to oxidative stress
csn-miR166 g-5p_2ss5GA8GT	Probable glutathione S-transferase	Unigene12682_All	4	337–356	Microtubule cytoskeleton organization
csn-miR167k_R-3_1ss9CT	Auxin response factor 5-like	CL6842.Contig1_All	4	927–946	Regulation of transcription
	Auxin-responsive factor AUX/IAA-related isoform 1	CL1028.Contig5_All	4	2632–2651	Regulation of transcription
	Auxin response factor 7-like	CL3788.Contig2_All	1	1554–1574	Regulation of transcription
csn-miR169(a/q)_R-2, csn-miR169b, csn-miR169e_1ss21GC, csn-miR169e_R-3, csn-miR169e_R-3, csn-miR169 g-p3_1ss10CT, csn-miR169i-p3_1ss6 GA, csn-miR169x_R + 1_2ss11TC21AC	Nuclear transcription factor Y subunit A-3	CL531.Contig_All	3	1183–1202	Regulation of transcription
csn-miR396b-p3/p5_2ss18AT19CT, csn-miR396b-p3_2ss18AT19CT	Catalase isozyme 1	Unigene31213_All	4	3428–3445	Response to hydrogen peroxide
	Leucine-rich repeat protein kinase family protein	CL2998.Contig2_All	4	1693–1711	Protein phosphorylation
csn-miR398a	Putative cytochrome c oxidase subunit 5b-like	CL8327.Contig1_All	4	140–161	Transport response to oxidative stress
	Putative cytochrome c oxidase subunit 6b-like	CL8327.Contig3_All	4	140–161	Transport response to oxidative stress
csn-miR408_L-1 R + 1,csn-miR408-p3_2ss18GT19GT	L-ascorbate peroxidase	CL8183.Contig2_All	4	54–74	Response to oxidative stress
	Serine/threonine protein phosphatase	Unigene2009_All	3	706–726	Protein phosphorylation
csn-miR477 g-p5, csn-miR477 g-p5_1ss1CT, csnmiR477 g-p5_2ss1CT22CT	Phenylalanine ammonia-lyase	CL3059.Contig24_All	4	2027–2044	L-phenylalanine catabolic process
csn-miR482b-p5_2ss3TA19TA	Calcium-dependent protein kinase 8-like	Unigene27192_All	2.5	740–757	Defence response
csn-miR482b-p5_2ss9TA19AC	Nbs-lrr resistance protein	CL11925.Contig1_All	2	724–742	Defence response
csn-miR821b-p5_2ss14TG20AT	Peroxidase 47	Unigene19710_All	4	512–530	Plant-type cell wall organization
csn-miR828a	Transcription factor MYB75	Unigene28964_All	2	654–675	Regulation of transcription
	R2r3-myb transcription factor	CL10500.Contig1_All	3.5	335–355	Regulation of transcription
	Transcription factor MYB114-like	CL7722.Contig1_All	3	348–369	Regulation of transcription
csn-miR858a_L-1 R + 1	Transcription factor MYB114-like	CL7722.Contig1_All	3.5	315–335	Regulation of transcription
	R2r3-myb transcription factor	CL10500.Contig1_All	3.5	335–355	Regulation of transcription
	Transcription factor MYB75	CL2876.Contig1_CKL	3.5	391–411	Regulation of transcription
csn-miR858b_R-3	Transcription factor MYB75-like	CL2876.Contig1_CKL	2	394–411	Regulation of transcription
csn-miR3633a-3p_R-3_1ss7TA	Nbs-lrr resistance protein	CL11925.Contig1_All	2	724–742	Defence response
csn-miR5368-p5_1ss(3/4)TC	Leucine-rich repeat protein kinase family protein	CL2998.Contig2_All	4	750–768	Protein phosphorylation
csn-miR5368-p5_2ss15TC18CT	Probable receptor-like protein kinase At5g39020-like	CL2968.Contig3_All	3.5	1194–1211	Development and cell growth
	Serine/threonine protein kinase isoform 1	CL1886.Contig3_All	4	321–338	Protein phosphorylation
csn-miR6173-p3_1ss4CT	Mitogen-activated protein kinase kinase kinase 1-like	Unigene9124_All	3	2373–2392	Protein localization to cilium
csn-miR6300-p3_1ss10AG	Transcription factor MYB75-like	Unigene21580_All	4	75–92	Regulation of transcription
csn-miR7733-p3/p5_2ss12CT17CA	Auxin response factor 9	CL2670.Contig1_All	4	100–118	Regulation of transcription
csn-miR8005a-p5_1ss11AG	Peroxidase 12-like	CL5005.Contig3_All	4	684–701	Response to oxidative stress
csn-miR8757b-p5_1ss1TG	Catalase isozyme 1	Unigene31213_All	4	3266–3283	Response to hydrogen peroxide
PC-3p-1581_1049	LRR receptor-like serine/threonine-protein kinase FLS2	CL5515.Contig3_All	4	891–910	Protein phosphorylation
PC-3p-70583_26	Serine/threonine-protein kinase PBS1-like	Unigene24032_All	4	1614–1634	Protein phosphorylation
PC-3p-106557_16	Serine/threonine-protein kinase SAPK2	CL6734.Contig1_All	3.5	73–92	Protein phosphorylation
PC-3p-134668_11	Calcium-dependent protein kinase	CL7018.Contig2_All	3.5	659–679	Abscisic acid-activated signalling
	Calmodulin-domain protein kinase 5 isoform 1	CL7130.Contig2_All	4	1785–1805	Abscisic acid-activated signalling
PC-3p-180347_8	Brassinosteroid insensitive 1-associated receptor kinase 1	Unigene4959_All	4	1256–1275	Signal transduction
	Peroxidase 24-like	CL4250.Contig2_All	4	679–697	Metal ion transport
	Calmodulin-like protein 3	Unigene14013_All	4	583–603	Mrna splicing, via spliceosome
PC-3p-184859_7	Peroxidase 24-like	Unigene24313_All	3	330–351	Metal ion transport
PC-3p-58653_32	Glutathione reductase	CL8717.Contig3_All	4	512–531	Glutathione metabolic process
PC-5p-167506_9	Mitogen-activated protein kinase homolog MMK1-like	CL6186.Contig1_All	4	1436–1457	Response to reactive oxygen species
PC-5p-80764_22	WRKY family transcription factor	CL1957.Contig1_All	3	778–797	Regulation of transcription

Figure 3. The hypothetical model of regulatory networks of Colletotrichum gloeosporioides–responsive miRNAs and their target genes in tea plant. Leucine-rich repeat protein kinase (LRR-PKs), serine/threonine protein kinase (Ser/Thr-PKs), LRR receptor-like Ser/Thr protein kinase (LRR-Ser/Thr-PKs), calcium-dependent protein kinase (CDPKs), mitogen-activated protein kinase (MAPKs), WRKY family transcription factor (WRKYs), NAC domain transcription factors (NACs), nuclear transcription factor Y (NF-YAs), auxin response factor (ARFs), Myb domain proteins (MYBs), nucleotide-binding site leucine-rich repeat (NBS-LRRs), catalases isozyme1 (CAT), L-ascorbate peroxidase (L-APX), glutathione reductase (GR), glutathione S-transferase (GST), cytochrome c oxidase (COX), peroxidase (POX), phenylalanine ammonia-lyase (PAL).

Figure 4. The general mechanism of miRNAs (A), amiRNAs (B) and target mimicry (C) in plants. RNA polymerase II (RNA Pol II), stem-loop precursor miRNAs (Pre-miRNAs), DICER-LIKE-1 RNase III endonucleases (DCL1), double-stranded RNA (dsRNA) binding protein, HYPONASTIC LEAVES1 (HYL1), s-adenosyl methionine-dependent methyltransferase HUA ENHANCER 1 (HEN1), argonaute family protein (AGO1).

Figure 5. Hypothetical model of miRNA identification and its strategies that target mRNA for the improvement of tea plant against biotic stress factors. Invading Ectropis oblique elicitor or Colletotrichum gloeosporioides pathogen can trigger the production of tea plant endogenous miRNAs that can target their respective mRNA for degradation were determined by HTS and degradome sequencing. Although tea plant-Ectropis oblique or Colletotrichum gloeosporioides derived miRNA-mediated gene silencing have not yet been discovered, we illustrate hypothetical model that identified miRNAs and target will be used to generate transgenic plants with miRNA-mediated gene silencing approaches, namely, over-expression of endogenous miRNA, artificial miRNA, miRNA mimic which will induce or suppress of the host and pathogen transcripts to generate biotic stress tolerance in tea plant.